SOMATIC HYBRIDIZATION

• Development of hybrid plants through the fusion of somatic protoplasts of two different plant species/varieties is called somatic hybridization.

Somatic hybridization technique

1. isolation of protoplast1. isolation of protoplast

2. Fusion of the protoplasts of desired species/varieties2. Fusion of the protoplasts of desired species/varieties

3. Identification and Selection of somatic hybrid cells3. Identification and Selection of somatic hybrid cells

4. Culture of the hybrid cells4. Culture of the hybrid cells

5. Regeneration of hybrid plants 5. Regeneration of hybrid plants

Protoplast• Protoplast also known as a naked plant cell refers to

all the components of plant cell excluding the cell wall.

• Protoplast is the biologically active and most significant material of cells.

• Plant cell wall acts as physical barrier and protects cytoplasm from microbial invasion and environmental stress.

• It consists of a complex mixture of cellulose, hemicellulose, pectin, lignin, lipids, protein,

• For dissolution of different components of the cell wall it is essential to have the respective enzymes. 

History

• Hanstein introduced the term ‘Protoplast’.

• The isolation of protoplasts from was first achieved through by Klercker (1892) on plasmolysed cells.

• Cooking (1960) for the first time isolated the protoplasts of plant tissues by using cell wall degrading enzymes viz., cellulase, hemicellulase, pectinase, and protease extracted from a saprophytic fungus Trichoderma viride & Myrothecium verrucaria.

• First achievement in protoplast fusion by Power (1970)

Isolation of Protoplast

(Separation of protoplasts from plant tissue))

1. Mechanical Method 2. Enzymatic Method

1. Mechanical Method

Plant Tissue

Collection of protoplasm

Cells Plasmolysis

Microscope Observation of cells

Cutting cell wall with knife Release of protoplasm

1. Mechanical Method

• Used for vacuolated cells like onion bulb scale, radish and beet root tissues

• Low yield of protoplast

• Laborious and tedious process

• Low protoplast viability

Enzymatic Method

Leaf sterlization, removal of epidermis

Plasmolysed cells

Plasmolysed cells

Pectinase +cellulase Pectinase

Protoplasm released Release of isolated cells

cellulase

Protoplasm released

Isolated Protoplasm

Enzymatic Method

• Used for variety of tissues and organs including leaves, petioles, fruits, roots, hypocotyls, stem, shoot apices, embryo, microspores.

• Mesophyll tissue - most suitable source

• High yield of protoplast

• Easy to perform

• More protoplast viability

Protoplast FusionProtoplast Fusion(Fusion of protoplasts of two different genomes)(Fusion of protoplasts of two different genomes)

1. Spontaneous Fusion 2. Induced Fusion

Intraspecific Intergeneric ElectrofusionMechanical Fusion

Chemofusion

Spontaneous Fusion

• Protoplast fuse spontaneously during isolation process mainly due to physical contact.

• Intraspecific produce homokaryones• Intergeneric have no importance

Intraspecific protoplast fusion

• Intraspecific protoplast fusion is the cross between the same species in an individual which involves the isogenic strains or the non-isogenic ones.

• The true value of protoplast fusion as a mean for establishing parasexual crosses has been realized so far in a few fungi. For example, in Cephalosporium acremonium.

• This technique offers the only way of carrying out crosses  and genetic analysis.

•

• Protoplast fusion technique made it possible to produce a preliminary genetic map of 8 linkage groups for C. acremonium. Genes which enhance the production of antibiotics have been identified and allied to specific linkage groups.

• The other examples are :

-Absidia glauca, Candida maltosa, Aspergillus niger, Fusarium graminearum, Penicillium verruculosum, T. reesei, etc. 

Interspecific protoplast fusion

• Interspecific protoplast fusion is the crosses between two different species.

• Interspecific protoplast fusions are of much importance in the area where new products are to be produced. Due to new genetic set up many noval secondary metabolites such as, antibiotics may be produced.

• Some of the examples where interspecific hybrids were produced through protoplast fusion are:

- S. cerevisiae x S. fermentali,

- S. cerevisiae x S. lipolytica,

- P. chrysogenum x P. notatum,

•  

Induced Fusion

• Chemofusion- fusion induced by chemicals

• Types of fusogens• PEG

• NaNo3

• Ca 2+ ions• Polyvinyl alcohol

Induced Fusion

• Mechanical Fusion- Physical fusion of protoplasts under microscope by using micromanipulator and perfusion micropipette.

• Electrofusion- Fusion induced by electrical stimulation

• Pearl chain of protoplasts is formed by low strength electric field (10kv m-1)

• Fusion of protoplasts of pearl chain is induced by the application of high strength electric field (100kv m-1) for few microsecond.

Protoplast fusion and somatic hybrids

– the fusion process

• electrofusion – protoplasts are aligned in a special chamber, electric current is applied, opening channels in cell membrane

• PEG fusion – protoplasts are coated with PEG, then incubated together; where cell membranes fuse, channels begin to form

• after fusion, "fusion products" begin to "round up"

Protoplast viability

• The most frequently used staining methods for assessing protoplast viability are:

- Fluorescein diacetate (FDA) staining

- Phenosafranine staining

- Calcofluor white (CFW) staining

Fluorescein diacetate (FDA) staining

• FDA, a dye that accumulates inside the plasma membrane of viable protoplasts.

• Viable intact protoplasts fluoresce Yellow green within 5 min.

• FDA is dissolved in CH3COCH3 & used at a concentration of 0.01%.

Phenosafranine staining

• It is specific for dead protoplasts that turn Red in staining procedure.

• Viable cells remain unstained by Phenosafranine

Calcofluor white (CFW) staining

• CFW binds to the β-lined glycosides in the newly synthesized cell wall which is observed as a ring of fluorescence around the plasma membrane.

Protoplast density

• Protoplasts have both maximum as wellas minimum plating densities for growth.

• Published procedures suggest that protoplasts should be cultured at a density of 5x103 to 106 cells/ml with an optimum of about 5x104 protoplasts/ml.

Identification and Selection of somatic hybrid cells

• Hybrid identification- Based on difference between the parental cells and hybrid cell with respect to

• Pigmentation • Cytoplasmic markers

• Fluorochromes like FITC (fluoroscein isothiocyanate) and RITC (Rhodamine isothiocyanate) are used for labelling of hybrid cells

• Presence of chloroplast• Nuclear staining

• Heterokaryon is stained by carbol-fuschin, aceto-carmine or aceto-orcein stain

Culture of the hybrid cells

• Hybrid cells are cultured on suitable medium provided with the appropriate culture conditions.

Regeneration of hybrid plants

• Plants are induced to regenerate from hybrid calli.

• These hybrid plants must be at least partially fertile, in addition to having some useful property, to be of any use in breeding schemes.

Advantages of somatic hybridization

• Production of novel interspecific and intergenic hybrid– Pomato (Hybrid of potato and tomato)

• Production of fertile diploids and polypoids from sexually sterile haploids, triploids and aneuploids

• Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance and many other quality characters.

Advantages of somatic hybridization

• Production of heterozygous lines in the single species which cannot be propagated by vegetative means

• Studies on the fate of plasma genes

• Production of unique hybrids of nucleus and cytoplasm

Limitations of Somatic hybridization

• Poor regeneration of hybrid plants• Non-viability of fused products• Not successful in all plants. • Production of unfavorable hybrids• Lack of an efficient method for selection of hybrids• No confirmation of expression of particular trait in

somatic hybrids

Application of Somatic hybridization

– protoplast fusion to create somatic hybrids

• "wide crosses" where even embryo culture won't work

–Citopsis gilletiana (wild) x Citrus sinensis

–citrus sexually incompatible spp.

–wild relative has disease/nematode resistance

–somatic hybrid used as a rootstock

– protoplast fusion to create somatic hybrids

• Solanum somatic hybrids

–S. tuberosum dihaploids fused with wild diploid S. chacoense

–resulting somatic hybrid (4n) is backcrossed to S. tuberosum cultivars (also 4n)

–overcomes sterility due to ploidy differences between somatic and sexual hybrids

Protoplast culture and regeneration

• From the protoplast solution of known density (about 105 protoplast/ml) about 1 ml suspension is poured on sterile and cooled down nutrient medium in Petri dishes.

• The plates are incubated at 25°C in a dim white light.

Protoplast culture and regeneration

• The protoplasts regenerate a cell wall, undergo cell division and form callus. The callus can also be subcultured.

• Embryogenesis begins from callus when it is placed on nutrient medium lacking mannitol and auxin. The embryo develops into seedlings and finally mature plants.